Colorectal cancer (CRC) is the second most common cause of cancer-related death in the USA and Europe. According to a recent review article by Pino, M. S. and Chung, D. D. in Expert Rev Mol Diagn, 10(5):651-665, 2010, CRC is responsible for an estimated 52,000 deaths per year in the USA and 146,000 per year in the EU. Approximately 2-5% of newly diagnosed cases of CRC can be attributed to Lynch syndrome.
Lynch syndrome (LS), often also referred to as hereditary non-polyposis colorectal cancer syndrome (HNPCC), is manifested by early-onset colorectal and endometrial cancer, and an increased risk of certain extracolonic cancers, including tumors elsewhere in the gastrointestinal tract (e.g., stomach, small bowel, biliary tract), urinary collecting system (renal pelvis, ureter) and the female reproductive system (ovaries).
Studies of selected LS families have estimated a 70-80% lifetime risk of colon cancer with a mean age at diagnosis in the mid-40 s. The second most common cancer in LS is endometrial cancer, and women with LS have a cumulative lifetime risk of developing endometrial and ovarian cancer, with a mean age at diagnosis approximately 10 years earlier than sporadic cases. The lifetime risk for gastric cancer in LS patients varies between populations, with a particularly high incidence in areas such as China and Korea, where there is a high endemic risk of gastric cancer, with a lifetime risk of approximately 30%. In these areas, the risk for gastric cancer exceeds that for endometrial cancer.
Lynch syndrome is highly associated with autosomal dominant inheritance of mutations in genes fundamental to the DNA mismatch repair (MMR) mechanism, and it is caused by germline mutations in one of four DNA mismatch repair (MMR) genes, MLH1, MSH2, MSH6 and PMS2. Moreover, 25% of sporadic colon tumours, as well as a number of tumours of endometrium, ovary and some other organs and tissues, are deficient in MMR. MMR proteins normally recognize and repair mismatched nucleotides and insertion/deletion loops caused by the slippage of DNA polymerase during replication of short repeat sequences known as microsatellites.
The five proteins involved in the human mismatch repair (MMR) mechanism to maintain genomic integrity function as heterodimers are MutLα (MLH1+PMS2), MutSα (MSH2+MSH6) and MutSβ3 (MSH2+MSH3). MMR proteins correct base/base mismatches and small insertion/deletion loops (IDLs) that arise on the newly synthesised strand during DNA replication and recombination.
The most frequently affected genes include MLH1, MSH2, MSH6 and PMS2, whose germline variations are reported in the LOVD database (www.insight-group.org/; www.lovd.nl/). Although, the majority of mutations affecting MMR genes are truncating, a significant proportion of mutations result in a single amino acid substitution or an in-frame deletion and are difficult to distinguish from harmless polymorphisms. Such alterations are often referred to as variants of uncertain significance (VUS) due to the uncharacterized effect of the variation on the function of the polypeptide.
MMR-deficient tumours are strongly associated with microsatellite instability (MSI). However, the degree and type of MSI differ depending on the MMR gene affected (Kantelinen et al., British J Cancer, 1-6, 2010).
The mean age of cancer onset in LS is significantly lower than that of sporadic colorectal cancer based on the fact that in LS, an individual has already inherited susceptibility through a mutated allele and only needs a second hit in a somatic cell to lose MMR activity and start tumorigenesis. Hence, LS tumors are characterized by the lack or lowered level of a causative MMR protein as well as impaired DNA repair causing MSI.
The timely recognition of LS is essential to identify patients at high-risk who will require intensive cancer surveillance. Colonoscopy screening and removal of precursor lesions, adenomas, significantly reduces cancer morbidity and mortality in MMR gene mutation carriers (Jarvinen et al., Gastroenterology 118: 829-834, 2000). Analyses of cost-effectiveness indicate that colorectal cancer surveillance in MMR gene mutation carriers is effective and considerably less costly than the consequences of no surveillance. The Centers for Disease Control and Prevention (CDC) in the United States recommends that all individuals with a new diagnosis of colorectal cancer, regardless of age or family history, should be offered genetic testing for Lynch syndrome. The CDC report further states that there is currently not enough evidence to recommend any specific screening strategy vs. alternative strategies.
However, the wide variety of clinical phenotypes complicates LS diagnostics and several clinical guidelines have been established to distinguish LS families from the general CRC burden. Currently, the clinical diagnosis of LS greatly relies on the Amsterdam criteria or the revised Bethesda guidelines, which take into account the age of cancer onset, the number and segregation of affected individuals in a family, and the level of MSI (Pino & Chung, Supra). However, many putative LS families do not fit these criteria and could be recognised as LS families only by characterizing a pathogenic germline MMR gene mutation in them.
The traditional diagnostic workflow to identify MMR gene mutation carriers, i.e., subjects afflicted by Lynch syndrome, involves several phases, such as tumor studies, DNA analyses, in vitro tests for mutation pathogenicity and proceeds from detecting a genetic defect to evaluating if it is associated with reduced MMR capacity. The first clinical step in diagnosing LS associated tumors includes immunohistochemistry (IHC) and MSI analysis followed by mutation screening dictated by the IHC and MSI results. One strategy for screening mutations is analyzing all four MMR genes (MLH1, MSH2, MSH6 and PMS2). When a pathogenic mutation is found, LS can be confirmed or in the absence of a MMR gene variation, considered unlikely but not ruled out (since no method, whether used alone or in combination, is 100% sensitive and specific).
A recent publication by the present inventors (Kansikas et al., Hum Mutat 32:107-115, 2011) discloses that testing in vitro synthesized mutant proteins for MMR capacity forms the cornerstone for pathogenicity testing. The published in vitro MMR assay studies the phenotypic consequences of LS mutations in a homologous human MMR system. Construction of the LS mutant protein requires that the genetic defect in question is found and fully characterized on DNA sequence level. A three-step decision tree was thus proposed (Couch et al., Hum Mutat 29:1314-1326, 2008):
However, there are several disadvantages associated with the present tests used for the establishment of a clinical diagnosis:
MSI testing is labour-intensive and requires expert molecular pathologic services, and while it is a hallmark for LS, it is not specific for it. Approximately 10-25% of sporadic CRCs and many extracolonic cancers also exhibit MSI. Furthermore LS-associated and sporadic MSI-positive CRCs have many histopathologic features in common, but differ in that sporadic MSI CRCs are not associated with a positive family history.
An inherent potential shortcoming of the IHC test is that the technique is somewhat subjective and depends upon the quality of tissue preparation, staining and interpretation of the results. Interestingly, abnormal staining patterns may be due to tissue preservation and the tumor microenvironment. For example, tissue hypoxia or oxidative stress may diminish the function of MMR proteins, even in genetically MMR-proficient tissues, leading to a focal loss or weak staining. Secondary abnormalities in MMR genes may also lead to rare staining patterns.
However, the most obvious limitation related to present methods of diagnosing LS, is that the tests are based on genetic testing and mutation analysis on subjects from a family with a known, or suspected, LS history, or on subjects already affected by cancer. This fact does not make present diagnostic methods suitable for routine screening of healthy subjects suspected of being carriers. Furthermore, the present testing schemes are very laborious requiring well-equipped laboratories as well as highly skilled laboratory personnel. It has been estimated that the steps including IHC, MSI and mutation analysis amount to about 3500 € per test, which is expensive for a routine test.
There is thus a well-established and recognized need for accurate, simpler, but cheaper and clinically suitable tests for identifying, whether a person is a carrier of MMR gene mutations related to Lynch syndrome and thus subjected to a high risk of colorectal cancer, as well as other cancers.